PVT1通过下调miR-194促进膀胱癌进展分子机制的研究

Previous studies confirmed miR-194 and PVT1 biological functions in bladder cancer , and miR-194 inhibited bladder cancer development through down-regulation PVT1 expression.We hypothesize that miR-194 and PVT1 played great roles in bladder cancer, but there is no report on miR-194 and PVT1 in MEDLINE and PUBMED databases ,and the mutual regulation relationship has not yet been fully revealed so that it need to be further in-depth study. In order to confirm the hypothesize,this project is proposed by constructing plasmid, genetic recombination and transfection, quantitative real-time PCR, Western Blot, luciferase activity detection, MTT, CCK-8 and flow cytometry analysis techniques and methods,and revealed PVT1 affect gene expression through miR-194, and revealed new bladder cancer gene regulatory pathways.PVT1 and miR-194 could become the new gene therapy targets for bladder cancer.Joint the intervention of both expression activities (down-regulation PVT1 and up-regulation miR-194) may be able to treat more effectively malignant phenotype bladder cancer, and to provide therapeutic direction for the accurate medical.

前期研究证实miR-194、PVT1在膀胱癌中的各自生物学功能，以及miR-194通过下调PVT1抑制膀胱癌的进展。我们推测miR-194、PVT1在膀胱癌中发生中具有重要作用,但在MEDLINE和PUBMED数据库中经文献检索，未见关于两者关系的报道,两者的相互调控关系尚未得到完全揭示，需要进一步深入研究。为证实此假设，本项目拟采用构建质粒、基因重组及转染、荧光定量PCR、Western Blot、luciferase活性检测、MTT或CCK-8、流式细胞分析等技术和方法，揭示PVT1通过miR-194影响的基因表达，揭示膀胱癌新的基因调控通路。PVT1和miR-194可能成为膀胱癌基因治疗的新靶点。联合干预两者的表达活性（下调PVT1和上调miR-194）或许能更有效地治疗恶性表型的膀胱癌，为精准医疗提供新的治疗方向。

长链非编码RNA在调控恶性肿瘤进展的过程发挥重要的作用。我们发现长链非编码RNAPVT1通过调节miR-194-5p和其他多个致癌基因调控膀胱癌的发生发展。PVT1通过扮演“miRNA分子海绵”的角色下调miR-194-5p的表达，并重新激活miR-194-5p致癌靶基因的调控通路，从而促进膀胱癌的进展。为进一步验证该假说，我们通过质粒构建、双素荧光酶等技术探究PVT1下调miR-194-5p的分子机制，发现PVT1是通过ceRNA机制抑制miR-194-5p的表达，其次通过siRNA,miRNAinhibition及mimic,过表达pcDNA3.1质粒等技术，发现PVT1通过抑制miR-194-5p促进膀胱癌细胞的增值、迁移并抑制其凋亡等；利用qRT-PCR和western-blot等实验技术，发现PVT1抑制miR-194-5p表达引起BCLAF1和WNT/β-catenin通路等蛋白表达升高。本项目揭示“PVT1-miR-194-5p-BCLAF1”轴在调控膀胱癌发生发展的重要作用，将来转化为一些膀胱癌潜在的诊断生物标志物和治疗靶点。